Exposure of cultured pituitary corticotrophs (AtT20-D16v, D16 cells) to phorbol esters causes a rapid increase in ACTH secretion which is followed by a refractory period (t 1/2 = 0.85 h). Desensitization appears to be due to uncoupling of PDBu receptor occupancy from activation of PKC and phosphorylation of substrates important for secretion. We will compare the biochemical properties of PDBu receptors and PKC from cytosols and membranes of control and desensitized cultures with respect to Ca2+ and phospholipid requirements, activation by PDBu, substrate specificity, phosphorylation state, subcellular localization by immunocytochemical techniques, and PKC isozyme content to determine if changes in PKC or isozyme composition may accompany desensitization. Our studies are greatly facilitated due to our success in producing PKC type-specific monoclonal and polyclonal antibodies. We will also determine if desensitization is associated with changes in levels of 2 inhibitory substances we have identified in these cells, loss of PKC substrates, or increased phosphatase activity for PKC substrates. Other agents, including Li+, Ca 2+, and glucocorticoids also cause desensitization to PDBu-directed ACTH secretion. The effects of these agents on the parameters listed above will be assessed to determine if they work through common or independent pathways. Prolonged treatment of D16 cells with PDBu causes loss of cellular PDBu receptors. This down-modulation also contributes to attenuation of PDBu- directed responses, but is clearly distinct from desensitization. We will determine whether down-modulation is associated with increased proteolysis of PKC, and whether this is PKC type-specific. We will characterize the biochemical and immunochemical properties of the PKC and PDBu binding activities remaining in down-modulated cultures to determine if they represent a specific portion of the total PDBu receptor population. The significance of these studies is due to the importance of PKC in regulation of cell growth and differentiated functions, as well as its role in tumorigenesis. Biochemical pathways associated with attenuation of PDBu-directed responses are likely to also play a role in limiting responses to hormones and growth factors whose mechanism of action involves PKC. Further understanding of biochemical pathways associated with limiting phorbol ester-directed responses may lead to pharmacological approaches for interfering with PKC activity.
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